1. Field of the Invention
The invention relates to the field of confocal laser scanning microscopy (CLSM) and in particular cardiac medical imaging.
2. Description of the Prior Art
Confocal laser scanning microscopy (CLSM) has emerged as a popular method for high resolution imaging of fluorescent labels, particularly in thick or scattering samples. By placing a pinhole in the conjugate optical plane, before the detector, out-of-focus light from above and below the focal plane is rejected from the image, enhancing the axial resolution. By collecting images from defined optical slices at successive depths, the three-dimensional arrangement of fluorescently-labeled structures can be derived. In traditional point scanning confocal systems, images are collected in a pixel-by-pixel manner and acquisition speeds for sequences with frame size 512×512 pixels are on the order of only a few frames per second.
Recent advances in beam shaping, the availability of fast CCD line detectors, and the implementation of efficient hardware for data transmission have made possible the development of a fast laser scanning microscope, the LSM 5 LIVE. A blade-shaped beam focused to a line (instead of a single point for conventional CLSM) permits the parallel acquisition of a whole line of pixels and reduces the scanning dimensionality to one direction. This microscope allows for the acquisition of two dimensional image-sequences (512×512 pixels) at frame rates of up to 120 frames per second (fps). This opens new avenues for a variety of fields.
In developmental biology, one major goal is to gain a better understanding of the mechanisms that influence the development of the cardiovascular system. In particular, it is desirable to assess the influence of genetic as well as epigenetic factors such as blood flow, heart wall forces, shear stress, etc. While the frame rates of typical confocal microscopes are suitable to study many dynamic processes occurring in living systems (e.g. cell migration, division, etc.), cell motions in the cardiovascular system (e.g. heart-wall motions, blood flow, etc.) typically occur at several millimeters per second, 2-3 orders of magnitude faster than cell migration. The significant improvement in frame rate offered by parallel scanning systems now makes it possible to collect image data from single optical sections of fast-moving structures.
However, resolving rapid three-dimensional motions in real-time still remains a challenge because it is not currently possible to scan the z-direction as fast as the xy plane. Other imaging modalities such as magnetic resonance imaging (MRI), computerized tomography (CT), or ultrasound (US) suffer from similar limitations. However, if the imaged body undergoes the same deformation at regular intervals and the acquisition is always triggered at a particular phase in the cycle, it is possible to assemble the data to recover a whole volume over one full period. For larger organisms (from mice to humans), it is relatively easy to gate the acquisition with respect to electrocardiograms (ECG) or respiratory signals, a technique known as prospective gating or triggering, and reconstruct volumes at a fixed moment in the cycle. Remaining motion artifacts may then be reduced by the use of various elastic registration procedures that warp the spatial data. In cases where gating is not possible or unreliable, nongated dynamic datasets have been registered by a variety of methods and for various purposes. For instance, in nuclear medicine, noise reduction may be performed through temporal averaging of nongated signals. For example, some researchers have used the imaging data from flow-encoded MRI to retrospectively perform the gating.
Using specific modifications to conventional MRI pulse sequences, it is also possible to generate and extract a signal which varies in synchrony with the cardiac cycle for later reconstruction. For CT, various methods have been developed, either to recover an imaged volume of the heart in a defined motion state at a single time point or for four dimensional imaging, by tracking the projection's center of mass. ECG-free algorithms have also been used for US imaging.
What is needed is an apparatus and method that make it possible to reconstruct dynamic three dimensional volumes of microscopic objects that are periodically moving, using currently available CLSM technology.